DESCRIPTION (from the application): The removal of introns from messenger RNA precursors by splicing is a mandatory step in the expression of most genes of higher eukaryotes. Recent work has defined a rare class of introns in most eukaryotic genomes that are spliced using an alternative pathway. Both the major and minor classes of introns are spliced via a similar mechanism in a large ribonucleoprotein structure known as the spliceosome. The two classes of introns are spliced in distinct spliceosomes which differ in RNA composition but nevertheless have a surprisingly high degree of similarity in the critical RNA-RNA interactions. In every case so far, minor class introns are found in genes which also contain multiple major class intron. This suggests that the two splicing systems must communicate with each other at the time of exon definition in order to insure the inclusion of exons flanked by introns of different classes. This application proposes to examine several aspects of splicing of this minor class of introns. First, elements of splice site communication will be examined by determining what sequences can potentiate the use of minor class splice sites in vivo. These experiments will use recently developed artificial alternatively spliced minigenes to test the function of selected sequences in splice site activation. Second, functional features of the small RNAs involved in splicing the minor class introns will be studied in vivo by swapping RNA domains between major and minor class small RNAs and between spliceosomal and self-splicing intron RNAs. This analysis will be extended to the analysis of function in vitro by developing an in vitro complementation system based on the in vivo demonstration of splice site mutant suppression in vivo. Third, the details of the sequence requirements of the minor class splice sites will be investigated further. Particular questions to be addressed concern how splice sites with apparent violations of consensus rules can be functionally spliced and whether splice sites exist in nature that can be used alternatively by either spliceosomal system. These investigations will also be extended to the in vitro analysis of spliceosomal complex formation on mutant pre-mRNAs to determine the step at which the splicing reaction is blocked.